The majority of intracellular proteins are phosphorylated at any given time, and, while nine of the 20 amino acids can be phosphorylated, the current focus has been on serine (Ser), threonine (Thr), and tyrosine (Tyr) phosphorylation despite pHis having been first identified over 50 years ago (Boyer, J. Biol. Chem., 3306 (1962)). These OH-containing amino acids form acid-stable, phosphoester (P—O) bonds upon phosphorylation (Attwood, et al., Amino acids 32, 145 (January 2007)). Histidine (His) forms a heat and acid-labile phosphoramidate (P—N) bond when phosphorylated. Phosphospecific antibodies have enabled the routine study of phosphoester protein phosphorylation, and the use of MS-proteomics has identified over 200,000 non-redundant sites of phosphorylation (Hornbeck et al., Nucl. acids res 40, D261 (January 2012)). The lack of specific antibodies to study pHis and the relative instability of the P—N bond under typical conditions used for proteomics have made it impossible to determine the prevalence of pHis, although it has been estimated that up to 6% of phosphorylation in eukaryotes occurs on His (Matthews, Pharmac. Ther. 67, 232 (1995)). Thus, it is possible that phosphohistidine (pHis) could be more abundant than phosphotyrosine (pTyr), which, despite its importance, comprises ˜1% of all known phosphorylation sites (Hunter and Sefton, Proc. Natl. Acad. Sci. USA 77, 1311 (Mar. 1, 1980, 1980); Olsen et al., Cell 127, 635 (Nov. 3, 2006)). Since current biochemical and proteomic technologies have been optimized for preservation, enrichment and detection of the phosphoester amino acids (pSer, pThr and pTyr), pHis has remained invisible.
pHis is unique among phosphoamino acids in that two distinct, biologically relevant isomers occur. The imidazole side chain of His contains two nitrogen atoms (N1 and N3) that can both be phosphorylated to generate two biochemically distinct isomers; 1-phosphohistidine (1-pHis) or 3-phosphohistidine (3-pHis) (FIG. 1A) which are also referred to as tele-phosphohistidine (τ-pHis) and pros-phosphohistidine (π-pHis) respectively (Attwood et al., Amino acids 32, 145 (January 2007); McAllister et al., Biochemical Society transactions 41, 1072 (August 2013)). NME1 and the closely related NME2 catalyze transfer of phosphate from ATP onto NDPs through a 1-pHis enzyme intermediate. The 3-pHis isomer has been shown to be more thermodynamically stable (Attwood et al., Amino acids 32, 145 (January 2007)) than 1-pHis and may be more prevalent. 3-pHis is used by bacterial histidine kinases that autophosphorylate to initiate phosphotransfer cascades and it also plays an important role as an enzymatic intermediate for phospholipase D as well as several key metabolic enzymes including; phosphoglycerate mutase (PGAM), succinyl-CoA synthetase (SCS), ATP-citrate lyase (ACLY) (see, for example, Bond et al., J. Biol. Chem. 276, 3247 (2001)).
There is a need for the development of specific, monoclonal antibodies (mAbs) for detection of pHis that can be used to detect and functionally evaluate novel sites of protein phosphorylation. These antibodies can be used, for example, to investigate signal transduction pathways.